The system of homologous recombination (HR) is responsible for the repair of DNA double-stranded breaks (DSB) and inter-strand cross-links (ICL), the most harmful DNA lesions. In yeast, Rad52 protein plays a key role in HR. In contrast, in mammals Rad52 knockouts are viable and show no distinct DNA repair and recombination phenotype. However, recently it was discovered that in mammals the function of RAD52 overlaps with that of BRCA2 and that inactivation of the RAD52 gene is lethal in human BRCA2-deficient cells (Feng et al., PNAS, 2011). Mutations in the BRCA2 gene are responsible for familial breast cancer that claims millions of lives. We will take advantage of this remarkable discovery by developing small-molecule inhibitors of RAD52 in order to selectively kill BRCA2-deficient breast cancer cells. The inhibitors will also present a useful tool for analysis of RAD52 function in the cell. In vitro, RAD52 promotes annealing of complementary ssDNA molecules. In order to identify inhibitors of the RAD52 DNA annealing activity by high throughput screening (HTS) we developed an in vitro FRET- based primary assay. The assay was validated in a pilot screen of the MLPCN compound library (Z' ~0.85) that yielded five putative RAD52 inhibitors (hits). Robust secondary and tertiary assays have been developed to evaluate the biological significance of these hits. To eliminate false positives due to fluorescence interference, the secondary assay will be used that employs DNA substrates with a pair of fluorophores that are different than in the primary assay. Additionally, an orthogonal assay using radioactively-labeled DNA substrates and gel-electrophoresis will be used to validate true hits. The specificity of the selected inhibitors will be examined using human RAD51 protein that is structurally unrelated to RAD52. The effect of confirmed RAD52 inhibitors on viability of BRCA2-deficient cells (Capan-1) will be tested. The Structure Activity Relationships (SAR) of the prioritized inhibitors will be developed to increase their selectivity and potency. In continuation of this grant, the mechanisms of RAD52 inhibition by the selected compounds will be investigated using several tertiary assays including RAD52 DNA binding, oligomerization, and ssDNA annealing. The therapeutic potential of the prioritized compounds will be examined using immuno-deficient mice with transplanted human xenografts.